1、BSI Standards Publication Ambient air Monitoring the effects of genetically modified organisms (GMO) Pollen monitoring Part 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-2-sampler PD CEN/TS 16817-1:2015National foreword This Published Document is the UK implementation of CEN/
2、TS 16817-1:2015. The UK participation in its preparation was entrusted by Technical Committee EH/2, Air quality, to Subcommittee EH/2/3, Ambient atmospheres. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to includ
3、e all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2015. Published by BSI Standards Limited 2015 ISBN 978 0 580 87461 1 ICS 07.080; 13.020.99 Compliance with a British Standard cannot confer immunity from legal obligatio
4、ns. This Published Document was published under the authority of the Standards Policy and Strategy Committee on 31 October 2015. Amendments/corrigenda issued since publication Date Text affected PUBLISHED DOCUMENT PD CEN/TS 16817-1:2015 TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZI
5、FIKATION CEN/TS 16817-1 O c t o b e r 2 0 1 5 ICS 07.080; 13.020.99 English Version Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen monitoring - Part 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-2-sampler Air ambiant - Surveillance des e
6、ffets dorganismes gntiquement modifis (OGM) - Surveillance du pollen - Partie 1 : chantillonnage technique du pollen laide dun filtre de masse pollen (PMF) et dun chantillonneur Sigma-2 Auenluft - Monitoring der Wirkungen von gentechnisch vernderten Organismen (GVO) - Pollenmonitoring - Teil 1: Tech
7、nische Pollensammlung mit Pollenmassenfilter (PMF) und Sigma-2-Sammler This Technical Specification (CEN/TS) was approved by CEN on 16 May 2015 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested
8、 to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at national level in an appropriate form. It
9、is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark
10、, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FO
11、R STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16817-1:2015 E PD CEN/TS 1681
12、7-1:2015CEN/TS 16817-1:2015 (E) 2 Contents Page European foreword . 5 Introduction 6 1 Scope 7 2 Normative references 7 3 Terms and definitions . 7 4 Basic principle of the procedure . 9 5 Sampling . 9 5.1 Instruments and materials 9 5.1.1 General 9 5.1.2 Sigma-2 passive sampler . 10 5.1.3 Pollen ma
13、ss filter PMF 10 5.2 Technical implementation 12 6 Sampling procedure 13 6.1 General . 13 6.2 Sampling design 13 6.2.1 General . 13 6.2.2 Exposure assessment of pollen input in the vicinity of fields with genetically modified crop (gm-fields) related to a specific GMO and region 14 6.2.3 Exposure as
14、sessment of pollen input for validating and/or calibrating dispersal models 15 6.2.4 General monitoring of pollen exposure at larger scales 15 6.2.5 Assessment of standardized and acceptor specific pollen deposition . 15 6.3 Site conditions . 15 6.4 Installing the equipment . 16 6.5 Exposure time 17
15、 6.6 Sampling at site . 17 6.6.1 Sigma-2 passive sampler . 17 6.6.2 PMF 17 6.7 Sample preparation . 18 6.7.1 Preparation of slides for microscopy 18 6.7.2 Preparation of PMF samples 19 7 Microscopic pollen analysis . 20 7.1 General . 20 7.2 Sigma-2 passive sampler . 20 7.2.1 Microscopic imaging meth
16、ods . 20 7.2.2 Qualitative analysis of the pollen diversity 21 7.2.3 Quantitative analysis of the pollen 21 7.3 PMF 21 7.3.1 Microscopic analysis . 21 7.3.2 Qualitative analysis of the pollen (diversity) . 21 7.3.3 Quantitative microscopic analysis of pollen 21 8 Molecular-biological analyses of GMO
17、 23 PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 3 9 Determination of the target parameters for GMO monitoring and representation of the results 24 9.1 General . 24 9.2 Sigma-2 passive sampler 24 9.2.1 Determination of pollen deposition per sampling period . 24 9.2.2 Determination of the daily mean
18、 pollen deposition rate per sampling period . 25 9.2.3 Determination of yearly pollen deposition . 25 9.2.4 References to pollen dispersal models . 25 9.3 PMF 25 9.3.1 Pollen count per sample N i,PMF25 9.3.2 Relative frequency of pollen species i . 26 9.3.3 Determination of pollen flux per sampling
19、period . 26 9.3.4 Determination of the daily mean pollen flux rate per sampling period . 26 9.3.5 Determination of the yearly pollen flux . 27 9.3.6 Assessment of results from molecular-biological analyses 27 9.3.7 References to pollen dispersal models . 28 10 Performance characteristics of the meth
20、ods 28 10.1 General . 28 10.2 Validation 28 10.3 Distribution of measured values . 29 10.4 Methodical approach and determination of basic parameters 29 10.5 Sigma-2 passive sampler 31 10.5.1 Sensitivity, detection limit and reproducibility 31 10.5.2 Detection confidence level and required numbers of
21、 cases . 33 10.6 PMF 35 10.6.1 Sensitivity, detection limit and reproducibility 35 10.6.2 Detection confidence level and required numbers of cases . 38 10.7 Parallel measurements. 40 10.8 Comparative measurements using a standard volumetric pollen trap (Hirst type) 42 10.9 Pollen diversity . 43 11 Q
22、uality assurance and quality control 44 11.1 General monitoring strategy and terms of reference of pollen monitoring with technical samplers . 44 11.2 Site protocol 44 11.3 Accompanying documentation for samples 45 11.4 Parallel measurements. 45 11.5 Comparative measurements using active samplers as
23、 calibration bases 45 11.6 Quality assurance and reference materials 45 11.7 Qualification . 46 Annex A (normative) Maize-specific requirements 47 A.1 Scope . 47 A.2 Basic principles . 47 A.3 Sampling 48 A.4 Sample preparation . 49 A.5 Quantitative microscopic pollen analysis 50 A.6 Molecular-biolog
24、ical analysis of maize DNA using PCR 51 A.6.1 General . 51 A.6.2 DNA extraction . 51 PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 4 A.6.3 Real-time PCR analysis . 51 A.7 Determination of the target parameters for GMO monitoring and assessment of the results . 52 Bibliography . 53 PD CEN/TS 16817-1:
25、2015CEN/TS 16817-1:2015 (E) 5 European foreword This document (CEN/TS 16817-1:2015) has been prepared by Technical Committee CEN/TC 264 “Air quality”, the secretariat of which is held by DIN. Attention is drawn to the possibility that some of the elements of this document may be the subject of paten
26、t rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. CEN/TS 16817, Ambient air Monitoring the effects of genetically modified organisms (GMO) Pollen monitoring, is composed of the following parts: Part 1: Technical pollen sampling using pollen mas
27、s filter (PMF) and Sigma-2-sampler the present document; Part 2: Biological pollen sampling using bee colonies. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bul
28、garia, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, T
29、urkey and the United Kingdom. PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 6 Introduction The European Parliament and the European Council require an environmental risk assessment and a post-marketing monitoring for any GMO released to the environment 5; 6. This had to be implied in national law in
30、 any member state of the EC by date. Pollen dispersal plays a significant role in the dissemination of genetically modified organisms (GMO). A procedure is described for GMO monitoring that enables quantification and documentation of GMO input and spread through pollen in a nationwide monitoring net
31、work which represents natural landscapes. Technical and biological pollen sampling (the present Technical Specification and CEN/TS 16817-2) and molecular biological analysis methods (polymerase chain reaction (PCR) for DNA; Enzyme-linked immunosorbent assay (ELISA) for proteins) are used for the det
32、ection of GMO input. It is reasonable to use both technical and biological sampling of pollen, thus they supplement each other in manifold ways. The technical sampling (i.e. the present document) is conducted with stationary point-samplers. They give a record of pollen input at the sample site that
33、correlates with the prevailing wind direction and relative position to the surrounding pollen sources. Bee colonies actively roam an area and are therefore area related samplers. Further, pollen sampling depends here on the collection activity of the bees and the availability of pollen sources withi
34、n the roaming area according to the bees preferences and supply of melliferous plants 32. Presently known pollen traps are only partially suited for GMO monitoring, since they can neither be standardized nor is the instrumentation designed for exposure times that are suitable for this purpose. Anoth
35、er limitation of commonly used pollen samplers is the requirement for a power supply, e.g. as for the Hirst type trap. The use of these instruments is therefore restricted to a limited exposure area. For these reasons, a new type of passive pollen sampler, the pollen mass filter (PMF), was developed
36、. The PMF is used either in combination with the Sigma-2 passive sampler or solely. The present Technical Specification is largely based on German VDI/Guideline 4330 Part 3 31. PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 7 1 Scope This Technical Specification describes a procedure for the use of t
37、he passive samplers Sigma-2 and PMF to sample airborne pollen. Both are designed to sample coarse aerosol particles. Collected samples are used to analyse pollen input with regard to pollen type and amount, and input of transgenic pollen. The Sigma-2 passive sampler here provides a standardized samp
38、ling method for direct microscopic pollen analysis and quantifying the input of airborne pollen at the site. The PMF yields sufficient amounts of pollen to additionally carry out molecular-biological diagnostics for detection of GMO. Essential background information on performing GMO monitoring is g
39、iven in VDI/Guideline 4330 Part 1 4, which is based on an integrated assessment of temporal and spatial variation of GMO cultivation (sources of GMO), the exposure in the environment and biological/ecological effects. Ideally, the pollen sampling using technical samplers for GMO monitoring should be
40、 undertaken in combination with the biological collection of pollen by bees (CEN/TS 16817-2). The application of technical passive samplers and the use of honey bee colonies as active biological collectors complement each other in a manifold way when monitoring the exposure to GMO pollen. Technical
41、samplers provide results regarding the pollen input at the sampling site in a representative way, whereas with biological sampling by honey bee colonies, pollen from flowering plants in the area is collected according to the bees collection activity. Thus, this method represents GMO exposure to roam
42、ing insects. By combining the two sampling methods these two main principles of exposure are represented. Furthermore, a broad range of pollen species is covered. The sample design depends on the intended sampling objective. Some examples are given in 6.2. 2 Normative references The following docume
43、nts, in whole or in part, are normatively referenced in this document are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. VDI 2119:2013-06 1) , Ambient a
44、ir measurements Sampling of atmospheric particles 2,5 m on an acceptor surface using the Sigma-2 passive sampler Characterisation by optical microscopy and calculation of number settling rate and mass concentration 3 Terms and definitions For the purposes of this document, the following terms and de
45、finitions apply. 3.1 acceptor surface natural or manmade collection surface for airborne particles 3.2 concentration number concentration number of particles per unit air volume; here number of pollen per m 3air 3.3 deposition pollen deposition deposition of atmospheric particles; here pollen on an
46、acceptor surface 1) For application of the Sigma-2. PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 8 3.4 dispersal pollen dispersal spread of pollen from the flower/field into the surrounding environment by wind drift 3.5 event unique DNA recombination event that took place in one plant cell, which w
47、as then used to generate entire transgenic plants 3.6 flux horizontal flux number of particles (here pollen) that are drifted horizontally per wind 3.7 genetically modified organism GMO organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or na
48、tural recombination SOURCE: Directive 2001/18/EC 5, modified The content of the definition was changed. 3.8 monitoring environmental monitoring characterizing the state and quality of the environment and its changes by measurements/observations in regard to defined objectives 3.9 pollen male gametop
49、hyte of the flowering plant 3.10 pollen type species class of pollen being distinguished by microscopic means on species, family or other order level 3.11 sampler device for sampling here of pollen 3.12 sampling pollen sampling collection of particles, here pollen by technical or biological means 3.13 sedimentation directed particle movement by gravity (here pollen in the air), which consists in a vertical flux towards the ground PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 9 4 Basic principle of the procedure For the technical
